The genus Linum L. (Linaceae) has more than 200 species. These species have agricultural, industrial, and medicinal importance. Domestication and continuous selection reduce genetic diversity of plants and in order to develop new and improved varieties, we should utilize all available genetic diversity in that crop and its wild relatives. The cultivated flax (L. usitatissimum L.) is an important crop plant and is being evaluated as a crop platform for the production of bio-industrial and nutraceutical products. In present study, we studied 230 plants of 23 populations in four Linum species namely, pale flax (L. bienne Mill.), cultivated flax (L. usitatissimum L.), and two wild relatives, L. austriacum L. and L. album L. The objective of this study was to investigate population genetic structure and genetic variability within each species using SSR and EST-SSR markers.The low degree of genetic variability was obtained in these species. AMOVA revealed significant genetic difference among species and populations. The significant correlation was observed between genetic and geographical distances the studied species. Evanno test and K-Means clustering revealed genetic fragmentation in the studied Linum species. Networking and STRUCTURE analysis revealed genetic affinity of the populations. The population assignment test revealed absence of gene flow or the occurrence of very limited gene flow among populations.